As described in the Wikipedia1 at http://en.wikipedia.org/wiki/Immunoassay: 1 The Wikipedia text quoted herein is released under CC-BY-SA, see http://creativecommons.org/licenses/by-sa/3.0.                “An immunoassay test is a biochemical test that measures the concentration of a substance in a biological liquid, typically serum or urine, using the reaction of an antibody or antibodies to its antigen. The assay takes advantage of the specific binding of an antibody to its antigen. Monoclonal antibodies are often used as they only usually bind to one site of a particular molecule, and therefore provide a more specific and accurate test, which is less easily confused by the presence of other molecules. The antibodies picked must have a high affinity for the antigen (if there is antigen available, a very high proportion of it must bind to the antibody).        Both the presence of antigen or antibodies can be measured. For instance, when seeking to detect the presence of an infection the concentration of antibody specific to that particular pathogen is measured. For measuring hormones such as insulin, the insulin acts as the antigen.        For numerical results, the response of the fluid being measured must be compared to standards of a known concentration. This is usually done though the plotting of a standard curve on a graph, the position of the curve at response of the unknown is then examined, and so the quantity of the unknown found.        Detecting the quantity of antibody or antigen can be achieved by a variety of methods. One of the most common is to label either the antigen or antibody. The label may consist of an enzyme, enzyme immunoassay (EIA)), radioisotopes such as 1-125 Radioimmunoassay (RIA), magnetic labels (magnetic immunoassay—MIA) or fluorescence. Other techniques include agglutination, nephelometry, turbidimetry and Western Blot. A number of these do form a directly visible line or test output but require an instrument to measure or capture the test output.        Immunoassays can be divided into those that involve labelled reagents and those which involve non-labelled reagents. Those which involve labelled reagents are divided into homogenous and heterogeneous (which require an extra step to remove unbound antibody or antigen from the site, usually using a solid phase reagent) immunoassays. Heterogeneous immunoassays can be competitive or non-competitive.                    In a competitive immunoassay, the antigen in the unknown sample competes with labelled antigen to bind with antibodies. The amount of labelled antigen bound to the antibody site is then measured. In this method, the response will be inversely proportional to the concentration of antigen in the unknown. This is because the greater the response, the less antigen in the unknown was available to compete with the labelled antigen.            In non-competitive immunoassays, also referred to as the “sandwich assay,” antigen in the unknown is bound to the antibody site, and then labelled antibody is bound to the antigen. The amount of labelled antibody on the site is then measured. Unlike the competitive method, the results of the non-competitive method will be directly proportional to the concentration of the antigen. This is because labelled antibody will not bind if the antigen is not present in the unknown sample.                        Because homogeneous assays do not require this step, they are typically faster and easier to perform.”        
As described in the Wikipedia1 at http://en.wikipedia.org/wiki/Lateral flow test: 1 The Wikipedia text quoted herein is released under CC-BY-SA, see http://creativecommons.org/licenses/by-sa/3.0.                “Lateral flow tests also known as Lateral Flow Immunochromatographic Assays are a simple device intended to detect the presence (or absence) of a target analyte in sample (matrix). Most commonly these tests are used for medical diagnostics either for home testing, point of care testing, or laboratory use. Often produced in a dipstick format, Lateral flow tests are a form of immunoassay in which the test sample flows along a solid substrate via capillary action. After the sample is applied to the test it encounters a coloured reagent which mixes with the sample and transits the substrate encountering lines or zones which have been pre-treated with an antibody or antigen. Depending upon the analytes present in the sample the coloured reagent can become bound at the test line or zone. Lateral Flow Tests can operate as either competitive or sandwich assays.        In principle any coloured particle can be used, however most tests commonly use either latex (blue colour) or nanometre sized particles of gold (red colour). The gold particles are red in colour due to localized surface Plasmon resonance. Fluorescent or magnetic labelled particles can also be used—however these require the use of an electronic reader to access the test result.        The sample first encounters coloured particles which are labelled with antibodies raised to the target analyte. The test line will also contain antibodies to the same target, although it may bind to a different epitope on the analyte.        The test line will show as a coloured band in positive samples.        The sample first encounters coloured particles which are labelled with the target analyte or an analogue. The test line contains antibodies to the target/its analogue. Unlabelled analyte in the sample will block the binding sites on the antibodies preventing uptake of the coloured particles.        The test line will show as a coloured band in negative samples.        Most tests are intended to operate on a purely qualitative basis. However it is possible to measure the intensity of the test line to determine the quantity of analyte in the sample. Implementing a Magnetic immunoassay (MIA) in the lateral flow test form also allows for getting a quantified result.        While not strictly necessary, most tests will incorporate a second line which contains an antibody that picks up free latex/gold in order to confirm the test has operated correctly . . .        Time to obtain the test result is a key driver for these products. Tests can take as little as a few minutes to develop. Generally there is a trade-off between time and sensitivity—so more sensitive tests may take longer to develop. The other key advantage of this format of test compared to other immunoassays is the simplicity of the test—typically requiring little or no sample or reagent preparation . . .        Probably the most well known examples of lateral flow tests are home pregnancy tests. However rapid tests or point of care tests are available for a wide range of applications including: HIV tests, Troponin T, test Malaria tests, drugs of Abuse tests, Fertility tests, Respiratory disease tests etc. Clinical tests can be applied to urine, saliva, blood, or stool samples. Tests are available for both human and animal diagnostics. Tests are also available for non clinical applications including testing food and water for contaminants.”        
FIG. 1 shows a typical prior art lateral or capillary flow pad assembly or strip as commonly used in rapid diagnostic applications. The strip contains an absorptive sample application or input pad 102, a conjugate pad 104, a membrane 106 along which the analyte flows, and a waste adsorbing pad 108. These components are bonded by an adhesive layer 110, onto a carrier strip 112, usually constructed from plastic sheet.
Immobilised on the membrane (typically nitrocellulose) are one or several test regions or line(s) 114 containing capture antigens or antibodies for the target(s) of interest, and a control region or line 116 containing a control capture antigen or antibody. As described above, visible or colored or fluorescent labels are incorporated, such that the test result is displayed as one or more visible or otherwise optically detectible lines at the test region(s) 114 and/or the control region 116.
Lateral flow strips such as that shown in FIG. 1 are often contained in a plastic cassette having an opening for sample introduction and a open or transparent “window” for viewing the test and control lines 114, 116.
Currently, lateral flow and other similar types of biomedical test strips are widely used to diagnose a range of medical conditions from pregnancy, health markers and infectious diseases, for example flu.
An immunoassay test device such as a lateral flow test strip lateral flow test strip or microfluidic cartridge can be broadly classified into one of two categories or types, depending on whether the immunoassay test device result can be determined by direct visual inspection of the test strip by a human user, (e.g., by reflectance or absorption), without requiring any test instrument or stimulating (e.g., optical) signal to determine the results of the test. For example, the test result of a pregnancy test strip can be viewed in the home by simple visual inspection under natural or otherwise ordinary ambient lighting conditions. For convenience of description, this general type of immunoassay test device is referred to herein as a “traditional” immunoassay test device, irrespective of its physical form.
Conversely, some other types of immunoassay test device (including those where fluorescent or magnetic labels are used) produce a test result that cannot be seen by ordinary or direct visual inspection, often requiring a stimulating signal and/or an appropriate type of sensor to detect the presence of the test lines on the strip (for example an optical signal of a specific wavelength or wavelength range, or a magnetic stimulating signal in order to stimulate the emission of a second signal from the test strip that is indicative of the test result). Furthermore, in some instances (e.g., where the label is fluorescent), the emitted second signal may be an optical signal technically visible to the eye of a user, but in a format that it outside the experience of most users such that can not be readily interpreted by a typical or inexpert user. For example, a traditional lateral flow strip result is readily assessed by an inexpert user, because it is in the form of dark absorptive lines in a red or blue or black colour against a light or white background (the membrane). However, a test result viewed under stimulation as described above might be visible only as feint lines on a dark background.
Additionally, the stimulated output can typically only be effectively imaged or sensed after the stimulating signal has been separated, for example after passing through a bandpass filter and associated optical components, and usually in the absence of ambient lighting. In other cases, the emitted second signal may not be an optical signal at all, requiring an appropriate type of sensor to detect the emitted second signal and processing of the detected signal to determine the test result.
The present invention particularly relates to this second broad class or type of immunoassay test device, where the test result is not visible to a human observer, at least without the assistance of a test instrument or apparatus. In particular, the inventor has identified a difficulty with these types of lateral flow test strips in which the test result is not directly visible to a user by simply viewing the lateral flow test strip under natural or ambient lighting. These types of tests require the use of an electronic reader instrument and/or at least specialised illumination to assess the result of the test. Currently, these test instruments or apparatus analyse the second signal emitted from the test strip in order to automatically assess the (binary) result of the test, displaying the results as a simple binary test result (e.g., “pass” or “fail”) or, at best, as a quantitative (numeric) value.
Although the lack of ambiguity in the test result provided by such an instrument may have direct application, the information that is available to the user is very limited. In particular, a simple binary test result does not provide any indication of the reliability of the assessment performed by the test instrument or apparatus, thus making it difficult for a user of the test instrument to have great confidence in the test result in the absence of any other information about the test result.
It is desired to address or alleviate one or more difficulties of the prior art, or at least to provide a useful alternative.